Received September 7, 2017; Revision received December 26, 2017
Abzymes with various catalytic activities are the earliest statistically
significant markers of existing and developing autoimmune diseases
(AIDs). Currently, schizophrenia (SCZD) is not considered to be a
typical AID. It was demonstrated recently that antibodies from SCZD
patients efficiently hydrolyze DNA and myelin basic protein. Here, we
showed for the first time that autoantibodies from 35 SCZD patients
efficiently hydrolyze RNA (cCMP > poly(C) > poly(A) > yeast
RNA) and analyzed site-specific hydrolysis of microRNAs involved in the
regulation of several genes in SCZD (miR-137, miR-9-5p, miR-219-2-3p,
and miR-219a-5p). All four microRNAs were cleaved by IgG preparations
(n = 21) from SCZD patients in a site-specific manner. The RNase
activity of the abzymes correlated with SCZD clinical parameters. The
data obtained showed that SCZD patients might display signs of typical
autoimmune processes associated with impaired functioning of microRNAs
resulting from their hydrolysis by the abzymes.
KEY WORDS: abzymes, schizophrenia patients, polyribonucleotide,
miRNA hydrolysis

Schizophrenia (SCZD) is a chronic brain disorder and one of the
highest-priority issues in psychiatry [1]. SCZD
prevalence reaches approximately 1%. It is the most severe psychiatric
disorder in humans that is associated with disintegration of thinking
and emotional responsiveness [2]. SCZD is a
progressive disorder with a polymorphic clinical picture; it often
results in impaired social adaptation and ability to perform work. SCZD
is accompanied with impairments in synaptic transmission that result in
the neuronal damage leading to severe brain dysfunctions [3-5] that often occur in
utero or in early childhood [6].

Despite the fact that SCZD has been studied for a long time, its
etiology and underlying pathogenetic mechanisms remain unclear. At the
moment, there is no common theory of SCZD pathogenesis. Opposing points
of view make it difficult to arrive to conclusions about pros or cons
of various theories and add little to the understanding of the role
that autoimmune response might play in SCZD [7, 8]. Nonetheless, a large body of evidence has
demonstrated a link between SCZD and various immunological processes
(see reviews [9-16]).

Analysis of single nucleotide polymorphisms in the genome-wide
association studies revealed a close link between the human leukocyte
antigen genes (HLA gene family), complement component 4, and other
immune system genes located on chromosome 6 (6p21-p22) in SCZD patients
[17-19]. Comparative
meta-analysis of 62 studies showed elevated levels of pro-inflammatory
cytokines in the blood serum and cerebrospinal fluid in a large cohort
of SCZD patients as compared to their healthy counterparts [20]. The association between inflammation and
schizophrenia was confirmed by the observed upregulation of the
Toll-like receptor-triggered production of cytokines by immune cells in
SCZD patients [21]. It was also found that
pro-inflammatory cytokines activate indoleamine 2,3-dioxygenase and
kynurenine monooxygenase in the tryptophan-kynurenine pathway, thereby
causing accumulation of 3-hydroxykynurenine and the NMDA antagonist
kynurenic acid [9], i.e., immunological disorders
might affect glutamatergic, serotonergic, dopaminergic, and
noradrenergic neurotransmission pathways [9-11].

Since 1980s, the ideas on the association between SCZD and autoimmune
processes have been published and widely debated, although not yet
commonly accepted. Impaired functioning of the immune system (including
autoimmune responses) and dysregulation of immune cells in SCZD
patients have been well documented [8, 13, 22-26].

Impairments in the central nervous system (CNS) in SCZD patients were
found to be related to autoimmune processes resulting in the
upregulated expression of autoantibodies against surface antigens of
the CNS cells, a neuropsychiatric disorder known as autoimmune
encephalopathy [22]. It is believed that the
damage of the brain cell membranes in SCZD patients results in the
exposure of self-antigens, and, as a consequence, production of
antigen-specific autoantibodies [23-25]. Interestingly, anti-glutamate receptor
antibodies were detected in patients with SCZD and other diseases,
including AIDs [26]. Antibodies against NMDA-NR1,
AMPA-GluR3, NMDA-NR2A, mGluR1, and mGluR5 were found in patients with
SCZD, but also in patients suffering from encephalitis, epilepsy,
systemic lupus erythematosus (SLE), neuropsychiatric SLE, cerebellar
ataxia, Sjogren’s syndrome, mania, and stroke. Such
anti-glutamate receptor autoantibodies can bind to neurons in several
brains regions, activate glutamate receptors and downregulate their
surface expression, activate endothelial cells of the blood–brain
barrier, cause brain injury, disturb glutamate signaling and related
functions, cause neuronal apoptosis, and induce psychiatric,
behavioral, and cognitive abnormalities [26].
Several epidemiological studies reported that SCZD is associated with
various AIDs. In particular, nine AIDs (thyrotoxicosis, celiac disease,
acquired hemolytic anemia, interstitial cystitis, Sjogren’s
syndrome, rheumatoid arthritis, type 1 diabetes, and myositis) have
higher prevalence in SCZD patients than in the comparison group; 12
AIDs were detected at higher prevalence in parents of SCZD patients
than in parents of SCZD patients [27]. An
association between SLE and SCZD was observed in patients of different
age, gender, and socioeconomic status [28]. SCZD
was also considered as atypical SLE with prevailing psychiatric
manifestations [29]. A subgroup of SCZD patients
was found to exhibit signs of multiple sclerosis (MS), both disorders
sharing similar etiology, while MS patients were discovered to have a
higher risk of developing SCZD [30]. In some
groups, association between SCZD and Hashimoto’s thyroiditis was
shown, as well as elevated levels of autoantibodies against
thyroglobulin and thyroid peroxidase [31-34]. Moreover, strong association between SCZD and
pemphigus [35] and the presence of
antiphospholipid antibodies in SCZD patients were documented [36]. Different subgroups of SCZD patients might
display signs of various well-known AIDs. Therefore, identification of
mechanisms underlying SCZD, including those involving autoimmune
factors, is very important.

It has been demonstrated by now that SCZD pathogenesis involves multiple
genes and their transcription products [37]. A
number of studies were aimed at elucidating the role of microRNAs that
are known to regulate as many as several hundreds of various genes [37-50]. Dysregulated expression
of various microRNAs has been found in the blood serum [38, 39], peripheral blood
mononuclear cells [40, 41],
and various brain regions [42, 43] of SCZD patients. Strong association between the
single nucleotide polymorphism in miR-137 and miR-9-5p and SCZD was
observed [17, 18, 44]. miR-137 was found to play an important role in
differentiation of embryonic stem cells, neuronal proliferation and
differentiation, and synapse maturation [45]. By
downregulating GluA1 subunit expression, miR-137 inhibits
AMPA-receptor-mediated synaptic transmission [46,
47], influences neurotransmitter release from
synaptic vesicles, and affects synaptic plasticity [48]. It also controls expression of the zinc finger
protein 804A (ZNF804A) gene. ZNF804A, in its turn, inhibits expression
of catechol-O-methyltransferase and dopamine receptor D2 [45], which results in impaired dopamine
neurotransmission. Moreover, dopamine receptor D2 expression is also
regulated by miR-9-5p [44]. miR-9-5p is involved
in neuronal migration; expression of miR-9-5p is lowered in neural
progenitor cells in SCZD patients [49]. miR-219 is
essential for neural stem cell proliferation and neurodevelopment [50], oligodendrocyte differentiation, and axonal
myelination [51].

Artificial catalytic antibodies (abzymes) raised against synthetic
analogs of transition states have been well described [52-54]. It was found that
natural autoantibodies can also display enzymatic activity. The
emergence of these antibodies in the blood serum is a typical feature
of various AIDs (see reviews [54-58]). Similarly to artificial abzymes [52-54], natural abzymes are
antibodies raised against haptens mimicking transition states of
catalytic reactions [54-58].
Moreover, anti-idiotypic antibodies against enzyme catalytic sites can
also exhibit catalytic activity (see [54-58] and in-text citations).

Despite the fact that autoantibodies against DNA, RNA, and various
proteins are found in the blood serum of healthy donors, such
antibodies often possess no catalytic activity [54-58]. It was shown that in
experimentally treated mice and patients with various AIDs, abzymes
with DNase, protease, and amylase activities can serve as the earliest
statistically significant markers of existing and developing autoimmune
pathologies [59, 60]. Abzyme
enzymatic activity might be detected even at the pre-disease stage, in
the absence of overt AID markers and changes in proteinuria, when
titers of antibodies against DNA, proteins, and other antigens still
fall within the normal range. Therefore, abzyme enzymatic activity may
be used as an indicative sign even at the pre-disease stage, as well as
during the development of spontaneous and induced AIDs [54-60].

It was mentioned above that SCZD does not belong to typical AIDs.
However, it was demonstrated recently that the blood serum from ~30%
SCZD patients contains high amounts of anti-DNA antibodies (as compared
to 37% in SLE patients); DNase [61] and
MBP-hydrolyzing [62] activities were detected for
the antibodies in the serum of 80-82% SCZD patients, thereby indicating
the existence of pronounced autoimmune component in SCZD
development.

Previously, we have detected RNA-hydrolyzing antibodies in the blood
samples from patients with SLE, MS, polyarthritis, and autoimmune
thyroiditis [63-66].

Considering the ability of catalytically active serum antibodies from
AID patients to hydrolyze DNA and RNA, association between microRNAs
and developing SCZD, and the role played by microRNAs in proliferation,
differentiation, and maturation of neuronal cells, here we examined the
RNA-hydrolyzing activity toward various RNA substrates and microRNAs of
antibodies isolated from the blood samples of SCZD patients.

MATERIALS AND METHODS

Materials. All proteins, reagents, and sorbents were from Sigma
or GE Healthcare (USA).

The patients in this study were diagnosed mostly with paranoid
schizophrenia (ICD-10 code – F20.0, 30 patients) and simple-type
schizophrenia (ICD-10 code – F20.6, five patients). Four out of
30 patients with paranoid schizophrenia were subtyped as F20.00
(paranoid), 13 as F20.01 (hebephrenic), 7 as F20.02 (catatonic), and 6
as F20.09 (unspecified).

Negative symptoms included loss or lack of normal personality traits and
human capabilities, e.g., depression, deficit of normal emotional
responses or other mental processes, poverty of speech, inability to
feel pleasure, no wish to form new relationships, and lack of
motivation. Such patients are less susceptible to medicated therapy [67, 70]. It is believed that
negative symptoms contribute more to the decrease in the quality of
life and social adaptation that the positive ones [71]. One patient with positive/negative symptoms was
found to have average parameters of positive and negative SCZD
symptoms. Diagnosis data are summarized in Table 1.

Table 1. General parameters for 35 patients
from three groups of SCZD patients

Note: Patients diagnosed with paranoid SCZD (ICD-10 code: F20.0; 30
patients) and simple SCZD (F20.6; 5 patients) were enrolled into the
study. Four patients were subtyped as F20.00 (paranoid), 13 as F20.01
(hebephrenic), 7 as F20.02 (catatonic), and 6 as F20.09 (unspecified).
PANSS scale allows standardized assessment of various psychopathology
symptoms in SCZD, to determine patient’s clinical profile, and to
follow up dynamic changes in the patient’s condition during
therapy. Each symptom is evaluated and presented as a score [48, 52].
PANSS+, scale for measuring positive (productive)
symptoms, such as delusions, mental disorders, hallucinations,
agitation, delusion of grandeur, suspicion, persecution mania, and
hostility. The symptoms are rated by comparing then to normal
psychological status. PANSS–, scale for
measuring negative symptoms, such as lowered emotional expression,
strangeness, communication difficulty, decrease initiative,
difficulties in abstract thinking, impaired spontaneous and fluid
speech, stereotyped thinking. The symptoms are rated by comparing then
to normal psychological status. Composite index is obtained by
subtracting the positive scale score from the negative scale score.
PANSS O, scale for general psychopathology syndromes, such as somatic
concerns (complaints of physical health problems), anxiety, feeling of
guilt, tension, mannerisms and posing, depression, motor retardation,
unsociable behavior, eccentric thoughts, disorientation, disturbance of
attention, disturbance of will, impulsivity, aggression, etc. General
SCZD severity is assessed. PANSS total, total score obtained by summing
up PANSS+, PANSS– and PANSS O.
* IgG preparations were tested for microRNA-hydrolyzing activity; the
assigned numbers are shown in the electrophoresis data (Figs. 2-5).# Average for 21 patients marked with asterisk in the
table.

The patients were diagnosed with SCZD in accordance with ICD-10, and
their diagnoses were clinically verified by the Department of
Endogenous Disorders, Mental Health Research Institute (Tomsk, Russia).
According to the provided information, all SCZD patients had a negative
history of typical systemic autoimmune and rheumatic diseases.

This study was approved by the Mental Health Research Institute Ethics
Committee, including patients’ written informed consent to use
blood samples for scientific research, in accordance with the
Declaration of Helsinki.

Antibody purification. Antibodies from the blood sera of 35 SCZD
patients and 10 healthy donors were purified and analyzed by the
earlier developed procedure [72-74] for purification of electrophoretically and
immunologically homogenous IgG preparations from the human blood serum.
The procedure included affinity chromatography of serum proteins on
Protein G-Sepharose followed by high-performance gel-filtration on a
Superdex-200 HR 10/30 column.

5′-Tagged miR-137 (5′-Flu-UUAUUGCUUAAGAAUACGCGUAG-3′),
miR-9-5p (5′-Flu-UCUUUGGUUAUCUAGCUGUAUGA-3′), miR-219-2-3p
(5′-Flu-AGAAUUGUGGCUGGACAUCUGU-3′), and miR-219a-5p
(5′-Flu-UGAUUGUCCAAACGCAAUUCU-3′) contained fluorescein
(Flu) at the 5′-end. Hydrolysis products were analyzed by
electrophoresis in denaturing 20% polyacrylamide gel (0.1 M Tris, 0.1 M
boric acid, 8 M urea, 0.02 M Na2EDTA, pH 8.3). The reaction
mixture containing 0.1 mg/ml IgG was incubated for 1 h. The gels were
analyzed using a Typhoon FLA 9500 laser scanner (GE Healthcare, USA).
The volume of blood samples and the amount of purified IgG antibodies
varied widely between the patients; therefore, experiments on microRNA
hydrolysis were performed only with 21 out of 35 IgG preparations. An
equimolar mixture of IgG preparations purified from the blood sera of
10 healthy donors (health-IgGmix) was used as a control.
Special care was taken to select volunteers who were fully free of
previous autoimmune, rheumatic, or allergic diseases, as well as viral
and bacterial infections, at least within 2-3 years prior to the
study.

Apparent Km and Vmax
(kcat) values were calculated based on the reaction
rate (V) dependence on the microRNA concentration using the
Microcal Origin v.5.0 software and presented as the double-reciprocal
Lineweaver–Burk plot [80]. The data obtained
were shown as a mean ± standard deviation based on three
independent experiments. The error of the estimations was less than
5-15%.

In situ analysis of the RNase activity for the equimolar mixture
(10 µg) of 35 IgG preparations from SCZD patients
(scz-IgGmix) was carried out in a denaturing 4-18% gradient
polyacrylamide gel (0.1% SDS) containing 40 µg/ml yeast RNA as
described earlier [75, 76].

Statistical analysis. The data were checked for the normal
distribution using the Shapiro–Wilk W test. The majority of the
parameters used for comparison did not fit the Gaussian distribution.
For this reason, we used the non-parametric Spearman’s rank
correlation coefficient test. In the case when the data fit the
Gaussian distribution, we used the parametric Pearson’s
correlation coefficient test was used. Differences between the samples
were evaluated using the Mann–Whitney U test. Significance was
set at p < 0.05. Median (M) and interquartile range (IQR)
were determined.

RESULTS

Patients. Thirty-five SCZD patients with different disease
symptoms according to the standard PANSS criteria [67-71] (Table 1) were divided into three groups: 16 – with
positive symptoms (patients 1p-16p), 18 – with negative symptoms
(patients 1n-18n), and 1 – with intermediate positive-negative
symptoms (patient 1 p/n). Major PANSS parameters characterizing each of
the groups are shown in Table 1.

Antibody purification. To examine the affinity and catalytic
activity of abzymes in the blood serum from 35 SCZD patients,
electrophoretically and immunologically homogeneous IgG preparations
were obtained as described earlier [63-65, 72-75].
It was found that after gel-filtration in acidic buffer (pH 2.6), the
maximal RNase activity coincided with the peak corresponding to
scz-IgGmix. Immobilized mouse IgG antibodies raised against
human IgG light chain fully absorbed this RNase activity.

Figure 1a shows the results of in situ
analysis of the RNase activity of IgG preparations after SDS-PAGE in a
gradient 4-18% gel containing yeast RNA. Hydrolyzed RNA (seen as dark
spots against fluorescent RNA background after gel staining with
ethidium bromide) was observed only for the scz-IgGmix
preparations. The fact that areas of RNA hydrolysis coincided with
zones corresponding to intact scz-IgGmix, together with the
lack of other areas of RNase activity (Fig. 1a),
proved that IgG preparations were free from contamination with canonic
RNases.

It has been shown before that IgG antibodies from the blood of healthy
donors do not possess RNase activity [63-66]. Figure 1 (b-d) shows typical
experimental curves for hydrolysis of poly(C), cCMP, yeast RNA, and
poly(A). Similar dependences were obtained for all the examined
substrates for all the 35 IgG preparations studied. The activities of
these preparations varied among the patients; however, all 35
preparations demonstrated detectable or high levels of RNase activity
(M/h per mg IgG) that declined in the order: cCMP (range, 0.3-1.7;
median/IQR, 1.06/0.29); poly(C) (0.1-1.2; 0.36/0.2); poly(A)
(0.09-0.50; 0.26/0.12); yeast RNA (0.04-0.48; 0.19/0.11).

The correlation coefficient (CC) for the parameters characterizing
hydrolysis of the examined substrates for the entire group of patients
was low except the CC value for cCMP and poly(C) hydrolysis (CC =
–0.599) (Table 2). Interestingly, no clear
correlation was observed between the RNase activity of 35 IgG
preparations and duration of the disease for any of the four substrates
(CC varied from –0.015 to +0.14). However, the level of cCMP
hydrolysis correlated with the patient age (CC = +0.369; Table 2). Moreover, the RNase activity toward cCMP
negatively correlated with the total PANSS score (CC = –0.45).

Table 2. Correlation between general
clinical parameters and enzymatic activity of antibodies from 35 SCZD
patients

Note: Non-parametric Spearman’s rank correlation coefficient test
was used except in cases of Gaussian distribution, when parametric
Pearson’s correlation coefficient test was used (marked with *).
The highest correlation coefficient values are shown in bold.

Thirty-five patients were divided into the three groups (Table 1). Mean RNase activities for IgG preparations from
patients with positive and negative symptoms differed insignificantly:
1.01-fold for cCMP, 1.01-fold for poly(C), 1.08-fold for poly(A), and
1.14-fold for yeast RNA. No significant difference was found between
the compared samples (Mann–Whitney test, p > 0.2).

For the entire group and patients with positive symptoms, the CC values
for the parameters of cCMP, poly(C), poly(A), and yeast RNA hydrolysis
were relatively low, either negative (from –0.056 to
–0.185) or positive (from +0.135 to +0.394). Significant
correlation was only found between the relative activities of cCMP and
poly(C) hydrolysis (CC = –0.791). Also, positive correlation was
observed between the level of cCMP hydrolysis and duration of the
disease, as well as patient age (CC = +0.540 and +0.753, respectively).
At the same time, the level of poly(C) hydrolysis negatively correlated
with the patient age (CC = –0.748), i.e., the RNase activity
decreased with age.

Similarly, the CC values for the IgG preparations from patients with
negative symptoms were rather moderate, either negative (from
–0.118 to –0.314) or positive (from +0.118 to +0.24).
Analysis of the RNase activity dependence on clinical parameters showed
that the level of yeast RNA hydrolysis correlated with the negative
symptom scale score (CC = +0.535) and composite PANSS index (CC =
–0.501).

MicroRNA hydrolysis. As mentioned above, some microRNAs regulate
up to several hundreds of genes in SCZD pathogenesis. Typical
hydrolysis patterns for miR-137, miR-9-5p, miR-219-2-3p and miR-219a-5p
including percentage of hydrolysis by each IgG preparation and mean
values for 21 IgG preparations are shown in Figs. 2-5 (for the correspondence between the numbers of
samples in the electrophoresis gels (1-21) and numbers assigned to
patients with positive and negative symptoms see Table 1). We found that health-IgGmix displayed
no activity toward the tested microRNAs.

Fig. 2. Hydrolysis patterns for Flu-miR-137 (0.01 mg/ml) cleaved
with IgG preparations (0.1 mg/ml) from the blood sera of 21 SCZD
patients. Hydrolysis products were detected from their fluorescence.
Panels (a) and (b) show antibody with number assigned, product length,
and percentage of site-specific RNA hydrolysis by each IgG preparation.
C, Flu-miR-137 incubation in the absence of antibodies; H, statistical
hydrolyzate of Flu-miR-137 (oligonucleotide length
markers).

Fig. 3. Hydrolysis patterns for Flu-miR-219a-2-3p (0.01 mg/ml)
cleaved with IgG preparations (0.1 mg/ml) from the blood sera of 21
SCZD patients. Hydrolysis products were detected from their
fluorescence. Panels (a) and (b) show antibody with number assigned,
product length, and percentage of site-specific RNA hydrolysis by each
IgG preparation. C, Flu-miR-219a-2-3p incubation in the absence of
antibodies; H, statistical hydrolyzate of Flu-miR-219a-2-3p
(oligonucleotide length markers).

Fig. 4. Hydrolysis patterns for Flu-miR-219a-5p (0.01 mg/ml)
cleaved with IgG preparations (0.1 mg/ml) from the blood sera of 21
SCZD patients. Hydrolysis products were detected from their
fluorescence. Panels (a) and (b) show antibody with number assigned,
product length, and percentage of site-specific RNA hydrolysis by each
IgG preparation. C, Flu-miR-219a-5p incubation in the absence of
antibodies; H, statistical hydrolyzate of Flu-miR-219a-5p
(oligonucleotide length markers).

Fig. 5. Hydrolysis patterns for Flu-miR-9-5p (0.01 mg/ml) cleaved
with IgG preparations (0.1 mg/ml) from the blood sera of 21 SCZD
patients. Hydrolysis products were detected from their fluorescence.
Panels (a) and (b) show antibody with number assigned, product length,
and percentage of site-specific RNA hydrolysis by each IgG preparation.
C, Flu-miR-9-5p incubation in the absence of antibodies; H, statistical
hydrolyzate of Flu-miR-9-5p (oligonucleotide length
markers).

The percentage of microRNA hydrolyzed by different IgG preparations
under similar conditions differed; the mean values declined in the
following order: miR-219a-5p (range, 7.4-99.7%; median/IQR, 88.4/46.6%)
≥ miR-137 (14.9-99.9%; 73.1/42.2%) ≥ miR-9-5p (3.1-99.9%;
57.5/64.2%) ≥ miR-219a-2-3p (7.4-99.7%; 53.2/67.7%) (Figs. 2-5). The correlation coefficients for the sets of
relative activities for three examined miRNAs (miR-9-5p, miR-219a-2-3p
and miR-219a-5p) were rather high (from +0.87 to +0.95). The CC values
for the correlation between the relative activities toward miR-137 and
three other miRNAs were relatively low and varied from +0.096 to +0.162
(Table 2).

It is known that in solution microRNAs adopt a hairpin structure. We
calculated the 3D minimal-energy hairpin structures for all the
microRNAs studied. The sites for the most active and moderate
hydrolysis in these microRNAs are shown in Fig. 6,
as well as average percentage of their hydrolysis by 21 IgG
preparations. It is evident that the three major cleavage sites for all
microRNAs are located in the loops or adjacent duplex regions. These
sites differ among the four microRNAs; however, in all of them, RNA
cleavage occurs either after or before G, although other possibilities
exist: 6G-7U, 13C-14G, and 8C-9C in miR-219a-5p; 5U-6G, 8U-9U, and
10A-11A in miR-137; 6G-7G, 8U-9U, and 13U-14A in miR-9-5p; 5U-6U,
8U-9G, and 13G-14G in miR-219a-2-3p (Fig. 6).

Fig. 6. Calculated 3D-structures for four microRNAs: miR-219a-5p
(a), miR-137 (b), miR-9-5p (c), and miR-219a-2-3p (d). The average
efficiency of microRNA hydrolysis at major and moderate cleavage sites
by 21 IgG preparations is shown.

Next, the microRNAs were denatured by heating up to 75°C followed by
quick cooling on ice. Representative hydrolysis patterns for miR-9-5p
and miR-137 by IgG-13n before and after denaturing clearly show that
the major hydrolysis sites in these microRNAs were preserved after
denaturation, although the relative amounts of hydrolysis products
slightly differed (Fig. 7a). The most important
difference was increased amounts of fragments 14 to 22 nucleotides in
length in the hydrolysis products of the denatured microRNAs. Similar
patterns were observed for the cleavage of miR-219-5p and miR-219a-2-3p
by IgG-13n and for all four microRNAs hydrolyzed by other IgG
preparations. Therefore, the cleavage of microRNA at the major sites
depends on the microRNA sequence rather than its unique structure.

Fig. 7. a) Hydrolysis of Flu-miR-9-5p and Flu-miR-137 (0.01
mg/ml) by IgG-13n (0.2 mg/ml) before and after miRNA denaturation.
Hydrolysis products were detected from their fluorescence after
reaction mixture incubation for 5-15 min; lane 15c corresponds
to microRNA before denaturation. b-d) Hydrolysis patterns for
Flu-r(pA)23 (b), Flu-r(pU)23 (c), and
Flu-r(pC)23 (d) cleaved with IgG preparations from different
SCZD patients at various incubation times. Numbers assigned to IgG
preparation are shown. Lanes C1 and C2 correspond to nondenatured and
denatured microRNAs, respectively, incubated in the absence of
antibodies; lane M, oligonucleotide length markers.

It seemed interesting to identify the sites of abzyme-catalyzed cleavage
in homo-oligonucleotides of similar length. Typical patterns for
r(pA)23, r(pU)23 and r(pC)23
hydrolysis by some of the IgG preparations are demonstrated in Fig. 7, b-d. In contrast to microRNAs, hydrolysis of
homo-oligonucleotides was less specific and occurred evenly along the
entire length of the homo-oligonucleotide, except more pronounced
cleavage between the 5´-terminal A4 and A5 nucleotides in
r(pA)23, U4 and U5 in r(pU)23, and C5 and C6 in
r(pC)23. These were the only major sites detected for all
the examined IgG preparations.

We also found significant correlation between the microRNA-hydrolyzing
activity and clinical parameters (according to Spearman’s
correlation coefficient test). Thus, the levels of miR-219a-2-3p and
miR-219a-5p hydrolysis by IgG preparations from SCZD patients
positively correlated with the PANSS general symptom score (CC = +0.479
and +0.583, respectively). The level of miR-219a-5p hydrolysis
positively correlated with the PANSS total score (CC = +0.524).

Analysis of correlation in the group of patients with positive SCZD
symptoms revealed that the level of miR-9-5p hydrolysis displayed a
strong negative correlation with the duration of the disease (CC =
–0.783), i.e., the microRNA-hydrolyzing activity declined with
age. A noticeable positive correlation was observed between the RNase
activity against miR-9-5p, miR-219-2-3p, and miR-219-5p and PANSS
general symptom score, as well as total PANSS score (CC > 0.75).

No significant correlation was found for the patients with negative
symptoms.

Effect of reaction conditions on RNA hydrolysis. The optimal
ionic strength for polynucleotide hydrolysis by scz-IgGmix
was determined for poly(C): the maximum hydrolysis was observed at 150
mM NaCl, i.e., under physiological conditions (Fig. 8a). After IgGmix was dialyzed against
EDTA, its relative activity increased ~5-fold (Fig. 8b). Further addition of MgCl2 and
MnCl2 resulted in the reaction inhibition, while addition of
Zn2+ ions to the dialyzed IgGmix strongly
activated RNA hydrolysis (Fig. 8b). It should be
noted that classic RNases are site-specific (e.g., RNase T1 selectively
cleaves RNA to the right of G bases, RNase A – to the right of
pyrimidines) [64-66].
Nuclease S1 selectively cleaves only single-stranded RNA fragments. The
abzymes from SCZD patients displayed none of these types of
specificity.

Fig. 8. The dependence of the efficiency of
IgGmix-catalyzed hydrolysis of poly(C) on the ionic strength
(a) and metal ion concentration (b). The dependence of the efficiency
of IgGmix-catalyzed hydrolysis of miR-137 on the reaction
mixture pH (c) and the presence of 10 mM EDTA and 40 mM metal ions
(d).

The activity of classic human RNases does not depend on the presence of
metal ions [81]. At the same time, the activity of
IgG antibodies derived from the blood serum of AID patients may either
depend on metal ions or be unaffected by them [64-66]. Previously, it was shown
that the purification procedure used in our study results in antibody
preparations containing small amounts of metal ions bound to the
antibodies [82]. The maximum miR-137 hydrolysis by
scz-IgGmix was observed in the absence of any added
components. The presence of 10 mM EDTA in the reaction mixture resulted
in approximately 1.7-fold decrease in the RNase activity.
Mn2+ and Zn2+ ions reduced the activity
~1.8-fold, whereas Mg2+ reduced it only 1.2-fold (Fig. 8). It is interesting that simultaneous presence of
EDTA and Mg2+ or Mn2+ ions in the reaction
mixture inhibited the RNase activity more profoundly (5.2-9.3 times)
than each of these agents separately. The combination of EDTA and
Zn2+ ions reduced the activity only 1.6-fold.

Abzymes from SLE and MS patients were found to display novel RNase
activity stimulated by Mg2+ [64-66]. An example of such activity is hydrolysis of
human mitochondrial tRNALys and its A50G mutant version by
abzymes from SLE patients [64-66]. When these tRNAs were hydrolyzed with RNase A
and other RNases traditionally used to analyze structural variations,
no differences in the cleavage patterns were observed [64-66]. However, when the same
molecules were cleaved with the abzymes in the presence of
Mg2+ ions, different hydrolysis products were obtained,
which indicated formation of new cleavage sites in the mutant molecule
and suggested local structural or conformational changes in the mutant
molecule [64-66]. In contrast
to the abzymes from SLE and MS patients, Mg2+ ions inhibited
rather than activated RNA hydrolysis by antibodies from SCZD patients
(Fig. 8, b and d). After dialysis, abzymes from
SCZD patients displayed the maximum activity in the presence of
Zn2+ ions (Fig. 8, b and d). Therefore,
it cannot be ruled out that a small fraction of abzymes from SCZD
patients are Zn2+-dependent RNases.

The pH optimum for miR-137 hydrolysis by scz-IgGmix was close
to pH 7.5 (Fig. 8c).

Previously, we have demonstrated that antibodies from healthy donors do
not hydrolyze RNA and DNA [64-66]. However, numerous published studies have
demonstrated the manifestations of autoimmune responses in SCZD
patients. SCZD was found to be associated with AIDs in many patients
(see above; [8, 13, 22-36]). However, SCZD is not
yet considered a typical AID. At the same time, IgG antibodies with the
DNase, RNase, proteolytic, or amylase activities are the earliest
significant markers of AIDs [59, 60]. Antibodies from SCZD patients efficiently
hydrolyze DNA and MBP [61, 62].

Here, we demonstrated that IgG antibodies from SCZD patients have
intrinsic RNase activity. While IgG antibodies from 90-95% SLE and MS
patients efficiently hydrolyze DNA and MBP [55-58], such activity was documented only for IgG
antibodies from 80-82% SCZD patients [61, 62]. However, we found that all examined IgG
preparations (100%) from SCZD patients efficiently hydrolyzed cCMP,
various homo-oligonucleotides, and microRNAs. Except for cCMP and
poly(C) (CC = –0.599), no significant correlation was observed
for the IgG-catalyzed hydrolysis of various substrates (Table 2); although correlation with some SCZD clinical
parameters was found.

Earlier, we were able to generate RNA-hydrolyzing abzymes by vaccination
of rabbits with DNA, RNA, DNase I, DNase II, and pancreatic RNase [60, 75]. Hence, it is possible
that the blood serum of patients with different pathologies contains
abzymes specific toward different RNAs. We demonstrated that all four
microRNAs examined were hydrolyzed in a site-specific manner (Figs. 2-5). However, the fact that the major hydrolysis
products for all homo-r(pN)23 were 4- to 6-nucleotide-long
oligonucleotides (Fig. 7, b-d) deserves special
attention. All four examined microRNAs were also cleaved into short
fragments of similar length (Figs. 2-5, a-d). It
has been shown before that the abzyme active site is localized in its
light chain that strongly binds only to 4-5 nucleotides in an
oligonucleotide [83]. The contribution of other
nucleotides to the binding decreases, and the dependence of the binding
affinity on the oligonucleotide length reaches a plateau at n
≥ 8-9 regardless of the total oligonucleotide length. Therefore, it
might be assumed that the antibody light chains efficiently recognize
4-6 5′-terminal nucleotides in any RNA. This might be the reason
why abzymes hydrolyze RNA mostly with the formation of fragments 4-6
nucleotides in length. At the same time, the existence of longer, less
efficiently generated products, suggests that the light chains could
form complexes with regions that are positioned away from the
5′-termini in oligonucleotides and microRNAs. The presence of
minor, but nevertheless significant, sites of hydrolysis in microRNAs
(Figs. 2-5, a-d) suggests that specific RNAs could
bind to antibodies via alternative mechanisms. Moreover, it cannot be
ruled out that generation of 4- to 6-nucleotide-long oligonucleotides
is the result of binding and subsequent hydrolysis of any RNA
substrates by either sequence-specific or non-specific abzymes. Most
likely, generation of major microRNA hydrolysis products is due to
their cleavage by antibodies specific toward these RNAs.

Usually, antibody-dependent catalysis is characterized with
102-106 times lower kcat values
as compared to canonical enzymes (see [54-58] and in-text citations). This higher substrate
affinity of abzymes results in lower kcat values,
since increase in the affinity leads to prolonged lifetime of the
antibody–substrate complex, and, as a consequence, lower catalyst
turnover number. For abzymes from AID patients, kcat
values vary within the 10–6-40 min–1
range (see [54-58] and
in-text citations).

The apparent kcat values for the abzyme-mediated
microRNA hydrolysis were relatively high (0.083-0.17
min–1), although still lower by approximately one
order of magnitude than those observed for the blood serum antibodies
from SLE patients [76, 84].
Because the specific activity of the antibodies was calculated based on
the total IgG concentration, specific RNase activities of individual
monoclonal subfractions could be substantially higher than that of
intact polyclonal IgGs.

The percentage of SCZD patients with high or reliably identified RNase
activity (100%) is substantially higher than that of SCZD patients with
the DNase activity (~80%) [61]. This might be
related to the fact that the RNase activity of an antibody preparation
is usually 30-300 times higher than its DNase activity, depending on a
patient and disease course [39-42, 44, 47,
56, 57, 63, 68], so that the RNase
activity may be detected in preparations with the DNase activity that
is too low to be identified. It cannot be excluded that virtually all
SCZD patients, whose clinical parameters are close to those shown in
Table 1, might have in their blood serum
antibodies displaying RNase activity.

Here, we demonstrated for the first time that polyclonal IgG antibodies
purified from the blood serum of SCZD patients possess RNase activity.
The presence of abzymes with the RNase, DNase, and proteolytic
activities suggest the development of autoimmune processes in SCZD
patients. MicroRNA-hydrolyzing abzymes might interfere with the
transcription of the corresponding genes and biosynthesis of their
protein products.

Recent studies performed at London Medical Institute Oliver House
support the theory that SCZD is a consequence of autoimmune brain
disorder [85]. As mentioned above, abzymes are the
earliest markers of the developing autoimmune response. In mice, the
pre-disease stages of spontaneous and antigen-triggered SLE and EAE
(model of experimental allergic encephalomyelitis or MS) are associated
with the changes in the differentiation profile of bone marrow stem
cells. These changes become even more pronounced with further
development of the pathological process [59, 86-89] and are related to the
emergence of abzymes hydrolyzing DNA, MBP, ATP, and polysaccharides in
the mouse blood serum. The activity toward DNA, MBP, and
polysaccharides of antibodies in the cerebrospinal fluid of MS patients
was 30-60 times higher (depending on the substrate type) than in the
blood of the same patients [90-92]. It is possible that the autoimmune disease
development starts in the cerebrospinal fluid and the brain, which is
in a good agreement with the theory [85] that SCZD
results from the impairments in the brain immune system.

Acknowledgments

We are thankful to A. V. Semke and N. A. Bokhan for kindly providing
blood samples collected from schizophrenia patients and to A. G.
Venyaminova and M. A. Vorobyeva for synthesizing specific
oligonucleotide probes and assisting in performing experiments.

This study was supported by the Russian Science Foundation (project
16-15-10103). Collection of blood serum samples from schizophrenia
patients and purification with partial characterization of antibodies
were supported by the SubProgram 1 of the Integrated Program of the
Siberian Branch of the Russian Academy of Sciences (III.2P.1/VI.57-5,
0309-2015-0022) and the Russian Foundation for Basic Research (project
16-04-00603).